PROJECT SUMMARY/ ABSTRACT Transfer RNAs (tRNAs) are the universal adaptor molecules necessary to convert the nucleic acid-based genetic code to protein sequence during protein synthesis (translation) by the ribosome. This process is universally conserved and fundamental to all life, and, as such, defects in the molecular players of translation, including tRNAs, result in diverse human diseases. Specific chemical modifications such as methylation are common in tRNA, but a detailed understanding of the enzymes that incorporate them and their contributions to tRNA function (and disfunction in disease) have only recently emerged for a few select examples. Since the discovery of the tRNA methyltransferase (Trm10) in Saccharomyces cerevisiae, an accumulating body of evidence, including phenotypes in yeast and a multisymptomatic disease associated with human mutations, has established a significant role for Trm10 in tRNA biology. To better understand the implications of Trm10 modification, the mechanism by which Trm10 recognizes and acts on tRNA needs to be addressed. This project aims to determine the molecular basis for Trm10 mechanism and function using a multi-disciplinary approach. Genetic, biochemical and molecular enzymology approaches will be combined with structural analyses of enzyme-tRNA complexes, and synthetic analogs of the native methyl donor, S-adenosyl-L-methionine, to uniquely identify the role of Trm10 in the maintenance of a high quality pool of tRNA. The studies will be performed in three complementary but independent aims that will: 1) Determine the molecular mechanism of methylation by Trm10, using biophysical and x-ray crystallographic structural analysis enabled by a novel mechanism (SAM analog)-based approach to trap enzyme-tRNA complexes, and complemented by biochemical analyses of Trm10 variants and studies to identify alternative substrates for Trm10 enzymes, cellular localization and native modification status; 2) Identify the molecular basis for tRNA substrate-selectivity of yeast and human Trm10 orthologs through detailed consideration of tRNA structure and stability; and, 3) Assess the roles of m1G9 in Trm10 target tRNAs in yeast and the zebrafish vertebrate model. Collectively, the proposed studies will advance the fields of enzymology, RNA biochemistry and tRNA biology by providing mechanistic and biological insight into a tRNA modification enzyme that is universally conserved among eukaryotes and critically important for human biology, yet whose molecular mechanism and biological functions are not at all understood. These results will also provide new insight into the dynamic landscape of tRNA modifications in multicellular eukaryotes.